Fluorescent proteins (FPs), in particular green fluorescent proteins, are commonly used fluorescent makers in molecular biology to monitor gene expression and protein localization in living organisms and in medical diagnostic applications.
Fluorescent proteins are found in a variety of marine organisms ranging from the jellyfish Aequorea victoria, to the Indo-Pacific coral Discosoma. Due to their genetically encoded fluorescence, fluorescent proteins have become most important marker molecules and tools in cell biology. Becoming spontaneously fluorescent without any requirements for cofactors, substrates or other gene products, FPs have revolutionized research in many areas of biology.
During recent years FPs have also gathered strong appreciation as powerful tools for the drug discovery process. As fluorescent probes, FPs enable both real-time and non-invasive reporting in living cells. This ability provides a basis for cell-based monitoring of FP-linked targets upon administration of external drugs. The impact of FPs has been revolutionary; FPs have not only facilitated visualization of intricate cellular architecture but they have also acted as markers of protein dynamics and behavior in cell biology. These applications have been translated to drug discovery where fluorescence proteins have been utilized in fluorescence and confocal imaging, HTS/HCS screening assays and for in vivo diagnostics. FPs cannot only be used in early stage target characterization but also in retrieving non-invasive ‘whole organism’ data and in evaluating lead compound toxicology.
Limitations of most fluorescent proteins are generally associated with molecular brightness and/or stability. Moreover, many FPs have additional complications involving protein folding, chromophore maturation and self-association. Although FPs have vastly improved over the years, mainly by introducing mutations, they still exhibit major limitations.
There is an interest in obtaining new fluorescent proteins with different properties compared to known fluorescent proteins. For instance, there are no fluorescent proteins on the market that can be used in paraffin sections at room temperature for immunohistochemical purposes since they lose their fluorescence intensity under such conditions.